Heatstroke-induced late-onset neurological deficits in mice caused by white matter demyelination, Purkinje cell degeneration, and synaptic impairment in the cerebellum

Global warming increases heatstroke incidence. After heatstroke, patients exhibit neurological symptoms, suggesting cerebellar damage. However, the potential long-term adverse outcomes are poorly understood. We studied the cerebellum after heatstroke in mouse heatstroke models. In this study, motor coordination disorder significantly appeared 3 weeks after heatstroke and gradually improved to some extent. Although white matter demyelination was detected at 1 and 3 weeks after heatstroke in the cerebellum, it was not found in the corpus callosum. The Purkinje cell numbers significantly decreased at 1, 3, and 9 weeks after heatstroke. The intensity of synaptophysin and postsynaptic density-95 temporarily appeared to attenuate at 3 weeks after heatstroke; however, both appeared to intensify at 9 weeks after heatstroke. Motor coordination loss occurred a few weeks after heatstroke and recovered to some extent. Late-onset motor impairment was suggested to be caused by cerebellar dysfunctions morphologically assessed by myelin staining of cerebellar white matter and immunostaining of Purkinje cells with pre- and postsynaptic markers. Purkinje cell number did not recover for 9 weeks; other factors, including motor coordination, partially recovered, probably by synaptic reconstruction, residual Purkinje cells, and other cerebellar white matter remyelination. These phenomena were associated with late-onset neurological deficits and recovery after heatstroke.

www.nature.com/scientificreports/ role in motor coordination/learning, sensory integration, and coordinate transformation. Cerebellar damage results in increased postural sway, hypermetric postural responses to perturbations and optokinetic stimuli, and postural responses that are poorly coordinated with volitional movement, which induces deficits in balance and walking 7 . After a heatstroke, patients who exhibit certain neurological symptoms-disorientation, wobbling (Supplementary Video S1), and vertigo-are bedridden, thereby preventing patients from returning to normal life, suggesting cerebellar damage. In addition to these neurological symptoms, several reports have revealed cerebellar abnormalities on head magnetic resonance imaging (MRI) in patients with heatstroke (Supplementary Fig. S1) 8,9 . Moreover, several interesting reports showed that neurological deficits appeared several weeks after heatstroke rather than immediately afterward 10,11 , and some cases improved with time 9 , while others are permanent. However, it is not well understood how these late-onset neurological deficits post heatstroke appear and, in some cases, improve over time. Therefore, we focused on the influence of heatstroke on the cerebellum using a mouse heatstroke model. Few studies have examined the CNS using a heatstroke model. Most CNS studies performed on animals after heatstroke have evaluated heat-induced cerebral ischemic injury 12 , hypothalamic injury 13 , and alcohol influence on the central amygdaloid nucleus 14 . These studies did not consider RH, and other heatstroke studies that did not examine the CNS also disregarded the RH impact and created desert-like conditions [15][16][17][18] . However, summer in temperate or tropical zones has high AT and RH. Even if AT remains the same, a higher RH accounts for a higher wet-bulb globe temperature (WBGT), suggesting a higher risk of heatstroke. Therefore, we recently established a mouse heatstroke model that accounts for AT and RH while monitoring WBGT. Our animal model presents low mortality (< 20%, 4 days after heatstroke) and is considered suitable for evaluating long-term adverse outcomes 19 . In this study, we examined late-onset motor coordination and cerebellar damage for 9 weeks in mouse heatstroke models.

Results
Motor coordination disorder appeared 3 weeks after heatstroke. Before heatstroke, the mean rotarod running time of mice in the control (CTL) and heatstroke (HS) groups was 85.6 ± 5.1 and 84.7 ± 5.2 s, respectively. Notably, 2 out of 36 mice in the HS group died within 1 day after heatstroke, and the remaining mice survived for 9 weeks. One week after heatstroke, the running time of the rotarod test in the HS group was 81.8 ± 6.8 s, whereas the running time in the CTL group was 93.1 ± 7.0 s. The running time of the rotarod test in the HS group 3 weeks after heatstroke further decreased to 68.8 ± 6.2 s and was significantly lower than that in the CTL group (92.7 ± 7.7 s, P < 0.05). The running time of the HS group gradually increased over time, whereas no differences were observed in the CTL group (Fig. 1a). To eliminate a feeding disorder after heatstroke, both groups of mice were weighed during the experiment, but these results were not statistically significant throughout the experiment (Fig. 1b).
Demyelination detected at 1 and 3 weeks after heatstroke in the cerebellar white matter. The cerebellar white matter in the CTL mice was dense and homogeneous to fast blue. However, the white matter in the HS mice was heterogeneous and sponge-like. Semi-quantification of myelin showed that the myelin percentage was significantly decreased at 1 (CTL, 70.4% ± 2.8%; HS, 59.7% ± 2.8%; P < 0.05) and 3 weeks (CTL, 69.3% ± 1.6%; HS, 63.8% ± 1.3%; P < 0.05) after heatstroke (Fig. 2), suggesting demyelination. However, this decrease was not statistically significant among the groups at 9 weeks, suggesting recovery.
In contrast, we compared the myelin quantity in the cerebellum with that in the corpus callosum for the same sections to determine region specificity. Interestingly, we found no significant differences between the CTL and HS groups during the experiments.
Expression of postsynaptic density-95 (PSD95) appeared to have temporarily attenuated at 3 weeks after heatstroke. We examined Purkinje cell synaptic plasticity based on PSD95 expression.
The distribution of PSD95-positive cells was strongly localized to the axon plexus. The PSD95-positive cells in the CTL group had a wide morphology, such as plates, but had low density. The PSD95-positive cells at 1 week after heatstroke were localized to the axon hillock of the Purkinje cells (looked similar to pen points). After 3 weeks, the immunoreactions appeared to have attenuated, although the calbindin-positive Purkinje cells were stained in the region. The positive reactions reappeared more heterogeneously at 9 weeks after heatstroke. Moreover, PSD95 immunoreactivity was observed again around the axon hillock of some residual Purkinje cells and appeared to have stronger intensities than that observed in the CTL group (Fig. 4). The co-staining of PSD95 with synaptophysin revealed that the intensity of synaptophysin immunoreactivity around the Purkinje cells appeared to have been the weakest at 3 weeks after heatstroke; however, both intensified at 9 weeks after heatstroke (Fig. 5).

Discussion
It is estimated that the recent global warming will cause an increase in patients with heatstroke in the near future. However, there is poor understanding of the potential long-term adverse outcomes on the CNS, although patients often exhibit neurological deficits 8 www.nature.com/scientificreports/ In this study, we evaluated the neurological deficits using the rotarod test and brain histological evaluations (in the cerebellum) for 9 weeks using a mouse heatstroke model because disappearing and swelling of the Purkinje cells have been reported in autopsied brain tissues of patients with heatstroke 21 . Observation of brain diffusionweighted MRI revealed hyperintensity in the bilateral cerebellar medullas in patients with heatstroke 22 . These reports indicate that cerebellar protection is a key therapeutic target in patients with heatstroke.
In our study, the running time of rotarod test was significantly shorter at 3 weeks after heatstroke and gradually returned to baseline at 9 weeks after heatstroke. These results suggest motor function/coordination impairment in our heatstroke model. In patients with multiple sclerosis, cerebellar ataxia is commonly observed, and diffuse white matter demyelination has been reported 23 . Therefore, we examined the morphology of the brain by Klüver-Barrera (KB) staining, but we could not recognize apparent cerebral infarction or hemorrhage. However, the cerebellum showed decreased intensity with KB staining and was recognized as sponge-like in the HS group. Semi-quantification of the KB-stained region revealed that the cerebellar white matter was significantly demyelinated between 1 and 3 weeks after heatstroke in the HS group. However, the intensity did not significantly differ from that of the CTL group at 9 weeks after heatstroke, suggesting remyelination. In contrast, demyelination could not be recognized in the corpus callosum during the experiments, and demyelination was cerebellar specific and might not have recovered until 9 weeks.
The calbindin-positive Purkinje cells in the CTL mice were aligned equidistantly on the Purkinje cell layer. The cell numbers had significantly decreased at 1 week after heatstroke and did not recover during the experimental periods, suggesting that the Purkinje cells degenerated. These results, including the rotarod test results, suggested that demyelination after heatstroke-induced cerebellar ataxia resulted in impaired motor coordination. These results resemble the outcomes observed in human patients with heatstroke 8,20-22 .  www.nature.com/scientificreports/ However, myelin recovered 9 weeks after heatstroke along with motor coordination, although the number of Purkinje cells did not recover during the experimental periods. To explain this discrepancy, we performed immunostaining of PSD95 and synaptophysin to examine synaptic plasticity and observed the Purkinje layer at a higher magnification. PSD95 was highly concentrated and tightly bound to the PSD of type 1 glutamatergic synapses, suggesting its critical role in protein assembly, synaptic development, and neural plasticity [23][24][25] . Synaptophysin is localized to the presynaptic vesicle membrane and contributes to membrane transport 26 . Its reduction has been considered to correlate with synaptic and neural impairments in Alzheimer's disease and traumatic brain injury [27][28][29][30] . The PSD95 immunoreactions were recognized in the same direction of the axon plexus surrounding the axon hillock of each Purkinje cell as previously reported 31 and were widely distributed, similar to plates or sheets, along with Purkinje cells. The degenerated Purkinje cells could not recover, and the cerebellar neuronal connections were temporally disrupted after heatstroke. However, the synapse and myelin staining results, including motor coordination, suggested that the surviving and residual Purkinje cells and the other cerebellar neurons were remyelinated and reconstructed new synaptic connections, probably inducing the recovery of partial motor coordination. It was suggested that these phenomena were associated with late-onset neurological deficits and recovery after heatstroke. www.nature.com/scientificreports/ The cerebellum exhibits diversity and dynamism 32 , and cerebellar dysfunction improved to some extent with time in our mouse heatstroke model, although there is a case that showed a permanent neurological deficit in human heatstroke. Synaptic impairment might not be improved if the Purkinje cell damages were severe after heatstroke. Our results indicated there is a time before demyelination or synaptic impairment. Therefore, some therapeutic interventions during this period may contribute to improving neurological deficit after heatstroke.
In this study, we neither analyzed skeletal muscle impairment nor determined the spinal cord impairment and the peripheral nerve fiber innervation, because of a circulatory failure due to massive hemorrhagic shock and ischemia-induced motor neuron necrosis in the spinal cord's ventral horn 33 . We did not systematically determine the presence of diffuse cerebral ischemia. Moreover, we did not perform electrophysiological examination and quantification of immunofluorescence staining (PSD95 and synaptophysin) to assess synaptic function after heatstroke. Further analysis is needed to clarify the heatstroke pathogeny and progression.

Conclusions
In the present study, we determined the motor coordination loss within a few weeks after the onset of heatstroke. Motor impairment was suggested to be caused by cerebellar dysfunctions that were morphologically assessed by myelin staining of cerebellar white matter and immunostaining of Purkinje cells with pre-and postsynaptic markers. However, although the Purkinje cell number did not recover for 9 weeks, other factors, including motor coordination, were partially recovered, probably by synaptic reconstruction, residual Purkinje cell, and other cerebellar white matter remyelination. It was possible that these phenomena were associated with late-onset neurological deficits and recovery after heatstroke. Further research is required to clarify the mechanism of cerebellar dysfunction in heatstroke to establish new therapeutic strategies.

Methods
Male C57/BL6J mice (age 10 weeks) were used in this study. All animals were purchased from SLC Japan Inc. (Shizuoka, Japan). The mice were allowed free access to food and water and were maintained on a 12-h light/dark cycle at room temperature (24 ± 2 °C) with constant humidity (40 ± 15%). All experimental procedures involving animals and clinical data were approved and overseen by the Institutional Animal Care and Use Committee of Showa University (#09022, 02003), which adhered to the ARRIVE guidelines. All methods were performed in accordance with the relevant guidelines and regulations.
Video of wobbling after human heatstroke and head MRI in patients with heatstroke were obtained from the participants. All experimental protocols were approved and overseen by the Clinical Trial Review Board of Showa University (F2019C83), which adhered to the CIOMS Ethical Guidelines for Biomedical Research. Informed consent was obtained from all participants and/or their legal guardians. Research has been performed in accordance with the principles of the Declaration of Helsinki. www.nature.com/scientificreports/ Protocol for heatstroke. The high AT and RH, resembling temperate/tropical summers, heatstroke model was used, which was based on our previous study 19 . A semi-enclosed heatstroke chamber (200 × 340 × 300 mm) made of acrylic was created by vertically stacking the animal cages in a greenhouse-like construction. An ultrasonic humidifier (USB-68, Sanwa, Japan) and a digital thermo-hygrometer (AD-5696; CA&D Company, Japan) were used for the humidification and monitoring of AT, RH, and WBGT. The heatstroke chamber was placed in an incubator (Bio-chamber, BCP-120F; TITEC, Japan) and preheated to the desired experimental temperature for ≥ 3 h. The humidifier was started 3 h before heatstroke to create a hot and humid environment. Meanwhile, the mice were allowed 3 h of water restriction, and the mildly dehydrated mice were placed in the heatstroke chamber and exposed to high AT (41 °C) and RH (> 99.0%) for 60 min. Subsequently, they were returned to the animal cage, where they could access food and water. Mice that were not heat-exposed were used as controls (CTL group).
Behavioral study (rotarod test). The rotarod test was performed according to a previous report 34 . A rotarod treadmill (Muromachi Kikai, Japan) consisted of a plastic rod (diameter, 3 cm; length, 10 cm) flanked by four large round plates (diameter, 57 cm). The rod rotates at a constant speed of 4 rpm at the beginning and continuously accelerates to a speed of 40 rpm for 5 min. The time each mouse spent on the rod was measured. Mice were trained with rotarod test once a week for five times before heatstroke. The behavior test in a mouse was performed twice within an interval of 5 min each and was expressed as the average of the trials. The animals were divided into two groups (HS and CTL) according to the scores of the last training (n = 36 in each group). The HS group mice were subjected to heat for 1 h. The CTL group (without HE) mice were prepared as a control. The HS and CTL groups were subjected to behavioral tests at 1, 3, 5, 7, and 9 weeks after heatstroke (Supplementary Fig. S2a).

Tissue preparation.
Under sodium pentobarbital (50 mg/kg, i.p.) anesthesia, the mice (HS, n = 9, per time course in each group) at 1, 3, and 9 weeks after heatstroke were transcardially perfused with 0.9% sodium chloride, followed by 10% neutralized formalin. The brain was removed and divided into two parts along the longitudinal cerebral fissure. Paraffin-embedded specimens of the right hemisphere of the brain were prepared. Subsequently, eight sagittal sections were sliced at a thickness of 5 μm at every 200 μm interval from the cerebral longitudinal fissure for histological examination, as described below. Age-matched CTL group mice were prepared to eliminate the influence of senescence ( Supplementary Fig. S2b).
Semi-quantification of cerebellar white matter demyelination. Cerebellar white matter demyelination was examined with the KB method without Nissl staining 33,35 . Eight sagittal sections of the cerebellar hemispheres were prepared at 200 µm intervals at the thickness 5 µm, stained with KB. These eight sections were tile scanned of the entire cerebellar hemisphere at 400 × magnification in each mouse using 9 animals per time course. The Luxol fast blue intensity, which stains myelin (fast blue) in the white matter, was then semiquantified to evaluate demyelination. These areas were manually traced with blinded investigators and converted to black and white; black areas were semi-quantified using Scion Image for Windows (Scion Corporation, USA). The pixels of the black myelinated regions were divided by the pixels from the total traced areas and expressed as percentages. Eight serials of sagittal sections were averaged for each animal. These procedures were performed by two investigators (H.Y. and K.Y.) who were blinded to the experimental groups.
Immunochemical staining and counting of Purkinje cells. Another serial series of eight sagittal sections were immunostained with antibodies against calbindin D-28k (calbindin) and used for counting Purkinje cells 36 . After removing the paraffin, using a series of xylene/alcohol solutions, the sections were incubated in 10 mM sodium citrate buffer (pH = 5.0) for 25 min to retrieve heat-induced antigen and immersed in 0.3% hydrogen peroxide/methanol for 30 min to block the endogenous peroxidase reaction. The sections were then incubated with a mouse Ig blocking reagent (M.O.M.; Vector, USA), followed by 5% goat serum wash to diminish the mouse endogenous immunoglobulin and non-specific reactions. The sections were then incubated overnight with a monoclonal mouse anti-calbindin antibody (1:2000; Swant, Switzerland) and incubated for 90 min with a biotinylated goat anti-mouse IgG the next day (1:200, DAKO, CA, USA). The immunoreactions were visualized by incubating with an avidin-biotin complex solution (Vector, USA) and diaminobenzidine (Sigma, USA). The calbindin-immunopositive Purkinje cells were determined and counted using the CellSens Standard software (Olympus, Japan). The number of Purkinje cells in the molecular layer of the cerebellum was counted twice manually in all lobules of the same section, and the average was calculated. Cell counting was repeated in the eight sagittal sections. This was also performed by an investigator (H.Y. and K.Y.) who was blinded to the experimental groups.
Immunostaining of synaptic markers. Post-and presynaptic markers were co-stained in the cerebellum at 1, 3, and 9 weeks after heatstroke to estimate the synapse around Purkinje cells using four of the nine mice. After heat-induced antigen retrieval and blocking according to the protocol as mentioned above, the sections were incubated with either monoclonal rabbit anti-calbindin antibody (